1. Field of the Invention
The present invention relates to methods of suppressing T cell activation, inhibiting CD44-mediated cell adhesion and CD44-monocyte IL1 release, treating inflammation, and transporting a drug or cytotoxic agent to a site of inflammation.
2. Background Information
Recent work has defined the importance of cell adhesion molecules in immune cell function (reviewed in Springer (1990) Nature 346:425-434; Haynes et al. (1989) Springer Sem. Immunopathol. 11:163-185; Hemler (1988) Immunol. Today 109-113). Cell adhesion molecules have been described that are receptors for soluble molecules (Haynes et al (1984) Nature 312:763-766), receptors for viruses (reviewed in Springer (1990) Nature 346:425-434; Dalgleish et al. (1984) Nature 312:763-766; Klatzmann et al. (1984) Science 225:59-63), and ligands for other cell surface molecules (reviewed in Springer (1990) Nature 346:425-434; Haynes et al. (1989) Springer Sem. Immunopathol. 11:163-185; Hemler (1988) Immunol. Today 109-113).
On immune cells, cell adhesion molecules mediate a wide variety of normal cell functions including cell movement, adherence to other cells, adherence to extracellular matrix proteins, mononuclear cell homing and monocyte cytokine release (reviewed in Springer (1990) Nature 346:425-434; Haynes et al. (1989) Springer Sem. Immunopathol. 11:163-185; Hemler (1988) Immunol. Today 109-113; Haynes et al. (1989) Immuno. Today 10:423-428). The CD44 molecule has been of recent interest because this protein has multiple proinflammatory functions, exists in soluble form in serum and plasma, and regulates the function of other adhesion molecules (reviewed in Haynes et al. (1989) Springer Sem. Immunopathol. 11:163-185; Haynes et al. (1989) Immuno. Today 10:423-428).
The CD44 molecule is an 85kd glycosylated molecule with N-terminal sequence homology to cartilage link proteins (Stamenkovic et al. (1989) Cell 56:1057-1062; Goldstein et al. (1989) Cell 56:1063-1072). Forms of CD44 of varying sizes have been described on many cell types (Haynes et al. (1989) Immunol. Today 10:423-428, Stamenkovic et al. (1989) Cell 56:1057-1062; Goldstein et al. (1989) Cell 56:1063-1072; Jalkanan et al. (1988) J. Immunol. 141:1615-1623). Variations in the size of CD44 isoforms have been suggested to be due to glycosylation differences, the addition of chondroitin sulfate molecules to CD44 (Jalkanan et al. (1988) J. Immunol. 141:1615-1623), and in some cases, to alternative splicing of CD44 mRNA (Dougherty et al. (1988) Exp. Hemat. 18:703, St. John et al. (1989) Req. Immunol. 300-310). Three forms of CD44 have been identified on peripheral blood mononuclear cells (PBMC) (Hale et al.) and an 85kd form (presumably a secreted form) has been identified in serum, plasma (Telen et al. (1983) J. Clin. Invest. 71:1878-1886; Lucas et al. (1989) Blood 73:596-600) and now synovial fluid.
CD44H is the hematopoietic form of the molecule, and additional forms are created by alternative splicing and insertion of up to 5 additional domains (Stamenkovic et al. EMBO J. (1991)10:343-348; Gunthert et al. Cell (1991) 65:13-24; Dougherty et al. J. Exp. Med. (1991) 174:1-5; Hoffman et al. Cancer Res. (1991) 51:5292-5297; Jackson et al. J. Biol. Chem. (1992) 267:1432-1439).
Functionally, the CD44 molecule has been shown to be a central molecule involved in T lymphocyte adhesion, T lymphocyte activation and monocyte cytokine release (Haynes et al. (1989) Immunol. Today 10:423-428; Jalkanen et al. (1986) Science 233:556-558; Jalkanen et al. (1987) J. Cell Biol. 983-990; Aruffo et al. (1990) Cell 61:1303-1313; Miyake et al. (1990) J. Exp. Med. 172:69-75; Lesley et al. (1990) Exp. Cell Res. 187:224-233; Stamenkovic et al. (1989) Cell 56:1057-1062; Goldstein et al. (1989) Cell 56:1063-1072; Jalkanan et al. (1988) J. Immunol. 141:1615-1623). The association of the CD44 intracellular domain with the cytoskeletal protein, ankyrin, and with the enzyme protein kinase C (PKC) (Kalomiris et al. (1989) J. Biol. Chem. 264:8113-8119) has suggested a role for CD44 in signal transduction of surface events to intracellular molecules. Ligand binding to the CD44 molecule promotes T cell adherence to monocytes via other adhesion molecule pathways (ICAM-1/LFA-1 and LFA-3/CD2) (Denning et al.; Koopman et al (1990) J. Immunol. 145:3589-3593) suggesting that CD44 can serve as a regulator of function of other adhesion molecules (reviewed in Haynes et al. (1989) Springer Sem. Immunopathol. 11:163-185; Haynes et al. (1989) Immunol. Today 109-113).
Recent studies have demonstrated that the CD44 protein is the primary receptor for hyaluronate in rodents and humans (Aruffo et al. (1990) Cell 61:1303-1313; Miyake et al. (1990) J. Exp. Med. 172:69-75; Lesley et al. (1990) Exp. Cell Res. 187:224-233). Both hyaluronate (Hiro et al. (1986) Biochem. Biophys. Res. Comm. 715-722) and CD44 mAB (Webb et al. Science, 249:1295) binding to monocytes induces monocyte IL1 release. On T cells, hyaluronate and CD44 mAB ligation of CD44 have disparate effects; CD44 mABs augment T cell triggering (Huet et al. (1989) J. Immunol. 798-801; Shimuzu et al. (1989) J. Immunol. 143:2457-2463) while hyaluronate suppresses T cell triggering (Anastassiades et al. (1984) Rheumatol. 11:734-729). Finally, CD44 mabs and polyclonal anti-CD44 serum have been shown to inhibit the binding of lymphocytes to high endothelial venules in inflammatory sites such as synovium (Jalkanen et al. (1986) Science 233:556-558; Jalkanen et al. (1987) J. Cell Biol. 983-990; Jalkanan et al. (1988) J. Immunol. 141:1615-1623), suggesting lymphocyte CD44 is one of several molecules involved in organ-specific lymphocyte homing. Thus, the hyaluronate receptor (CD44) molecule, by existing in several isoforms, and by virtue of wide cellular distribution, functional association with other adhesion molecules, and physical association with ankryin and PKC, is a multifunctional proinflammatory molecule involved in immune cell activation (reviewed in Haynes et al. (1989) Immunol. Today 10:423-428) as well as metastasis of certain tumor cell types (reviewed in Haynes et al. (1991) Cancer Cells 3:347-350).
Hyaluronate, the ligand for CD44, is an important component of synovial fluid and plays a critical role in maintaining high viscosity of synovial fluid in normal diarthroidal joints (reviewed in Schuber and Hammerman (1964) Bull. Rheum. Dis. 14:345-348; Castor et al. (1966) Arth. Rheum. 9:783-794). In rheumatoid arthritis (RA) synovial fluid, hyaluronate concentration and degree of polymerization is decreased (Castor et al. (1966) Arth. Rheum. 9:783-794). Reduction in synovial fluid hyaluronate concentration and degree of polymerization has been suggested to be an important factor leading to joint dysfunction and destruction in RA (Schubert et al. (1964) Bull. Rheum. Dis. 14:345-348; Castor et al. (1966) Arth. Rheum. 9:783-794), and potentially may decrease the immunosuppressive effect of hyaluronate on T cells (Anastassiades et al. (1984) Rheumatol. 11:734-729).
Applicants have demonstrated that CD44 is upregulated in RA on many synovial cell types and that the level of CD44 present in synovial tissue is directly proportional to the degree of synovial inflammation in RA. Applicants have also demonstrated that CD44 is immunosuppressive to T cells. The present invention relates, at least in part, to a method of interdiction of proinflammatory functions of the CD44 molecule.